γ干扰素对瘢痕疙瘩成纤维细胞转化生长因子β／Smad信号通路的作用  

Effects of interferon-gamma on the transforming growth factor beta/Smad pathway in keloid-derived fibroblasts

作　　者：刘佳琦[1] 胡大海[1] 张战凤[1] 官浩[1] 折涛[1] 张军[1] 白晓智[1] 

机构地区：[1]第四军医大学西京医院烧伤与皮肤外科,陕西西安710032

出　　处：《中华烧伤杂志》2009年第6期454-459,共6页Chinese Journal of Burns

基　　金：国家自然科学基金（30600644）.

摘　　要：目的了解γ干扰素（IFN-γ）对瘢痕疙瘩Fb（KFb）中TGF-β／Smad信号通路的作用，探讨IFN-γ治疗病理性瘢痕的可能机制。方法切取3例患者的瘢痕组织，体外分离培养KFb，实验选用第3-5代细胞。（1）将KFb分为：对照组，加无血清DMEM培养；TGF-β1组，用10ng／mL的TGF-β1单独作用；IFN-γ组，用100ng／mL的IFN-γ单独作用；TGF-1，＋IFN-γ组，10ng／mL的TGF-β1与100ng／mL的IFN-γ联合作用。采用实时荧光定量RT-PCR、蛋白质印迹法和免疫荧光细胞化学染色法，分别检测结缔组织生长因子（CTGF）的mRNA、蛋白表达，以及α平滑肌肌动蛋白（α-SMA）的蛋白表达与阳性细胞表达情况。（2）另取KFb，用10ng／mL的IFN-γ作用，于作用前及作用后30min和1、2、4、6、8h通过实时荧光定量RT-PCR检测Smad3和Smad7的mRNA表达，于作用前及作用后1、2、4、6、8h用蛋白质印迹法检测Smad3和Smad7的蛋白表达。（3）另取KFb，根据添加的IFN-γ终浓度不同分为1、10、100ng／mL IFN-γ组，均作用4h；设立未添加IFN-γ的KFb为对照组。同前检测各组Smad3和Smad7的mRNA及蛋白表达。结果（1）IFN-γ组KFb CTGF的mRNA和蛋白表达量为0.017±0.009与1.198±0.004，较对照组（0.024±0.013与1.229±0.011）显著减少（P〈0.05）；TGF-β1＋IFN-γ组CTGF的mRNA和蛋白表达量为0.634±0.138与1.204±0.010，较TGF-β1组（1.331±0.298与1.727±0.004）显著减少（P〈0.01）。IFN-γ组KFb中，α-SMA阳性细胞荧光强度（0.922±0.059）和α-SMA蛋白表达量（0.3051±0.0031）较对照组（1.055±0.005与0.4513±0.0094）显著减少（P〈0.01）；TGF-β1＋IFN-γ组-α-SMA阳性KFb荧光强度（1.129±0.004）和α-SNA蛋白表达量（0.6734±0.0098）较TGF-β1组（1.270±0.005与1.3842±0.0024）显著减少（P〈0.01）。（2）10ng／mL IFN-γ作用后第1个时相点，Smad3的mRNA和蛋白表达量均出现一过性增高，随后降低，mRNA表达量于作用后4h降至最Objective To elucidate the effects of interferon-gamma（IFN-γ） on the transforming growth factor beta （TGF-β）/Smad pathway in keloid-derived fibroblasts （KFb） , and to investigate the underlying mechanism in the treatment of pathologic scar with IFN-γ. Methods Keloid tissue of 3 patients were obtained, and then KFb were separated and cultured in vitro. KFb from passages 3 to 5 were used for the study. （1） KFb were divided into control group （incubated with serum-free DMEM）, TGF-β1 group （treated with 10 ng/mL TGF-β1 ）, IFN-γ group （ treated with 100 ng/mL IFN-γ）, and TGF-β1＋ IFN-γ group （incu- bated with 10 ng/mL TGF-β1 combined with 100 ng/mL IFN-γ）. The expression level of mRNA and protein of connective tissue growth factor （CTGF）, α smooth muscle actin （α-SMA） protein and expression of α-SMA positive KFb were detected by real-time fluorescent quantitation RT-PCR （FQ-RT-PCR） , Western blot and immunofluorescence cytochemical staining. （2） Another sample of KFb was obtained and treated with 10 ng/mL IFN-γ. The expression level of Smad 3 and Smad 7 protein was detected by Western blot before and 1, 2, 4, 6, 8 h post stimulation （PSH）. The expression level of Smad 3 and Smad 7 mRNA was assessed by FQ-RT-PCR before stimulation and 30 mins post stimulation and at PSH, 1, 2, 4, 6, 8. （3） Another sample of KFb was obtained and divided into 1, 10 and 100 ng/mL IFN-γ groups based on the con-centration of IFN-γ, treated for 4 hours; KFb without IFN-γ treatment was set up as control group. The ex- pression levels of the protein and mRNA of Smad 3 and Smad 7 were measured by FQ-RT-PCR and Western blot. Results （1） The level of mRNA and protein of CTGF in IFN-γ group （0. 017±0. 009 and 1. 198±0. 004） was respectively lower than that in control group （0. 024±0. 013 and 1. 229±0.011, P 〈 0.05）. The level of mRNA and protein of CTGF in TGF-β1＋ IFN-γ group （0. 634±0. 138 and 1. 204±0. 010） was respectively lower than that in

关 键 词：干扰素Ⅱ型 瘢痕疙瘩 成纤维细胞 转化生长因子β1 SMAD蛋白质类 

分 类 号：R686[医药卫生—骨科学]